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C T Amemiya

Publications and source records attributed to C T Amemiya.

At least 19 recordsLinked to original sources
PubMed2001

Generation of a P1 artificial chromosome library of the Southern pufferfish.

We describe the generation of a P1 artificial chromosome genomic library from the Southern pufferfish, Spheroides nephelus. The arrayed library consists of approximately 30,000 clones and has an average insert size of 125-150 kb. The coverage is estimated to encompass seven to eight genome equivalents. The library has been used for isolating numerous genomic clones and for establishing contigs of several multigene families. Analysis of several of the clones from this library suggests a preponderance of CA repeat tracts relative to their abundance in humans. The library and high-density filters have been made available to the scientific public through genomics distribution companies.

Animals↗
PubMed2001

Ikaros expression as a marker for lymphoid progenitors during zebrafish development.

The Ikaros gene encodes a transcription factor that, in mice, has been shown to be essential for the correct differentiation of B and T lymphocytes and is expressed in all cells of the lymphoid lineage, including pluripotent hematopoietic stem cells. During development in zebrafish, Ikaros expression begins in lateral mesoderm, and continues in the intermediate cell mass (ICM), which is derived from lateral mesoderm and has been shown to generate primitive hematopoietic precursors. Cells expressing Ikaros were then seen on the ventral side of the dorsal aorta, known to be a location of definitive hematopoietic precursors in birds and mammals. Ikaros-expressing cells were also found in the pharyngeal region, near the forming thymus. Later, such cells were seen in the pronephros, the site of hematopoiesis in adult fish. The timing of appearance of Ikaros-expressing cells suggests that, similar to other vertebrates, lymphocytes in the thymus arise from hematopoietic tissue located near the dorsal aorta or in the ICM.

Animals↗
PubMed2000

Evolution of Hoxa-11 in lineages phylogenetically positioned along the fin-limb transition.

HOXA11 is a transcription factor implicated in paired appendage development. To identify signatures of evolutionary change in the structural, and putative functional, domains of HOXA11, we studied its evolution in tetrapod and nontetrapod lineages that represent approximately 1.5 billion years of evolutionary time. Here, Hoxa-11 gene proper sequences were determined for frog (Xenopus tropicalis), coelacanth (Latimeria chalumnae), common zebrafish (Danio rerio; Hoxa-11a and Hoxa-11b paralogs), and giant zebrafish (D. aequipinnatus; Hoxa-11b) and aligned against previously published Hoxa-11 sequences of human, mouse, chick, and newt. Based on aligned Hoxa-11 amino acid sequences, the protein was demarcated into three segments: Domains I (N-terminal) and III (homeobox + C-terminal), which varied slightly in rates and patterns of evolution, and a variable, overall hydrophilic region (HyD), which partially overlaps with Domain I. As judged by character reconstructions of HOXA11 Domains I and III, no significant changes in rates of coding sequence evolution occurred in tetrapods (frog and chick), relative to coelacanth (a lobe-finned fish), i.e., across the fin-limb transition. Accelerated rates of Hoxa-11 coding sequence evolution were observed for the mammalian and newt lineages. This was shown to be a gene-specific phenomenon. The duplicated Hoxa-11a and Hoxa-11b genes of zebrafish exhibited accelerated rates of evolution and accumulated substitutions at sites that are conserved among coelacanth and all tetrapods examined. Amino acid sequence comparisons of the HyD of HOXA11 suggested that a putative repressor subdomain, containing stretches of consecutive alanine residues, emerged within the tetrapods. A high degree of nucleotide conservation in the 5' half of the Hoxa-11 intron was observed for tetrapod and nontetrapod lineages. Using electrophoretic mobility shift assays, a 35-bp intron sequence, which is 100% conserved in all Hoxa-11 loci except for the zebrafish Hoxa-11a paralog, was found to bind protein(s) in HeLa and chick whole-cell extracts.

Alanine↗
PubMed2000

A recombinogenic targeting method to modify large-inserts for cis-regulatory analysis in transgenic mice: construction and expression of a 100-kb, zebrafish Hoxa-11b-lacZ reporter gene.

The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions. In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice. PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting. A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting. Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f. transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites. These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones.

Animals↗
PubMed1999

A novel multigene family encodes diversified variable regions.

Antigen recognition in the adaptive immune response by Ig and T-cell antigen receptors (TCRs) is effected through patterned differences in the peptide sequence in the V regions. V-region specificity forms through genetically programmed rearrangement of individual, diversified segmental elements in single somatic cells. Other Ig superfamily members, including natural killer receptors that mediate cell-surface recognition, do not undergo segmental reorganization, and contain type-2 C (C2) domains, which are structurally distinct from the C1 domains found in Ig and TCR. Immunoreceptor tyrosine-based inhibitory motifs that transduce negative regulatory signals through the cell membrane are found in certain natural killer and other cell surface inhibitory receptors, but not in Ig and TCR. In this study, we employ a genomic approach by using the pufferfish (Spheroides nephelus) to characterize a nonrearranging novel immune-type receptor gene family. Twenty-six different nonrearranging genes, which each encode highly diversified V as well as a V-like C2 extracellular domain, a transmembrane region, and in most instances, an immunoreceptor tyrosine-based inhibitory motif-containing cytoplasmic tail, are identified in an approximately 113 kb P1 artificial chromosome insert. The presence in novel immune-type receptor genes of V regions that are related closely to those found in Ig and TCR as well as regulatory motifs that are characteristic of inhibitory receptors implies a heretofore unrecognized link between known receptors that mediate adaptive and innate immune functions.

Amino Acid Sequence↗
PubMed1999

Generation of a zebrafish P1 artificial chromosome library.

We have constructed a genomic P1 artificial chromosome library from the zebrafish. The library has been arrayed and archived in two hundred seventy-one 384-well microtiter dishes. It encompasses four to five genome equivalents with an average insert size of approximately 115 kb and is readily accessible to the scientific community. The library has been used by numerous investigators in the community and shown to be a useful reagent for chromosomal walking and positional cloning.

Animals↗
PubMed1999

Evolution of chordate hox gene clusters.

In this article, we consider the role of the Hox genes in chordate and vertebrate evolution from the viewpoints of molecular and developmental evolution. Models of Hox cluster duplication are considered with emphasis on a threefold duplication model. We also show that cluster duplication is consistent with a semiconservative model of duplication, where following duplication, one daughter cluster remains unmodified, while the other diverges and assumes a new architecture and presumably new functions. Evidence is reviewed, suggesting that Hox gene enhancers have played an important role in body plan evolution. Finally, we contrast the invertebrates and vertebrates in terms of genome and Hox cluster duplication which are present in the latter, but not the former. We question whether gene duplication has been important in vertebrates for the introduction of novel features such as limbs, a urogenital system, and specialized neuromuscular interactions.

Animals↗
PubMed1999

pPAC-ResQ: A yeast-bacterial shuttle vector for capturing inserts from P1 and PAC clones by recombinogenic targeted cloning.

We have developed a method to capture inserts from P1 and P1 artificial chromosome (PAC) clones into a yeast-bacteria shuttle vector by using recombinogenic targeting. We have engineered a vector, pPAC-ResQ, a derivative of pClasper, which was previously used to capture inserts from yeast artificial chromosome clones. pPAC-ResQ contains DNA fragments flanking the inserts in P1 and PAC vectors as recombinogenic ends. When linearized pPAC-ResQ vector and P1 or PAC DNA are cotransformed into yeast, recombination between the two leads to the transfer of inserts into pPAC-ResQ. pPAC-ResQ clones thus obtained can be further modified in yeast for functional analysis and shuttled to Escherichia coli to produce large quantities of cloned DNA. This approach provides a rapid method to modify P1/PAC clones for functional analysis.

Bacteria↗
PubMed1999

A long form of the skate IgX gene exhibits a striking resemblance to the new shark IgW and IgNARC genes.

Differential screening has been used to identify cDNAs encoding a long form of IgX in Raja eglanteria (clearnose skate). Comparisons of the IgX long form with the previously described short-form IgX cDNAs and the genomic IgX locus indicate that the V and two 5' C regions of the short and long forms of IgX are >90% identical at the nucleotide level. Differences between the V sequences of the long- and short-form IgX genes are concentrated in complementarity determining regions, suggesting that these forms are derived through alternative splicing of the same genomic loci or transcription of highly related loci. The extreme conservation of nucleotide sequence, including third position codons, among different cDNAs as well as the near identity of nucleotide sequence in the intervening sequences of germline IgX, IgX short-form sterile transcripts and IgX long-form sterile transcripts indicate that the multiple IgX loci are recently diverged from one another and/or are under intense gene correction. Phylogenetic analyses of the known cartilaginous fish immunoglobulin loci demonstrate that the long form of IgX is orthologous to IgW/IgNARC (NARC) and is most consistent with: 1) the divergence of the IgX/IgW/NARC and IgM-like loci from a common ancestral locus prior to the divergence of the cartilaginous/bony fish lineages and 2) the divergence of the NAR locus from the IgX/IgW/NARC gene(s) after the cartilaginous/bony fish split but prior to the shark/skate split, approximately 220 million years ago.

Amino Acid Sequence↗
PubMed1999

Zebrafish YAC, BAC, and PAC genomic libraries.

Numerous positional cloning projects directed at isolating genes responsible for the myriads of observed developmental defects in the zebrafish are anticipated in the very near future. In this chapter, we have reviewed the YAC, BAC, and PAC large-insert genomic resources available to the zebrafish community. We have discussed how these resources are screened and used in a positional cloning scheme and have pointed out frequently formidable logistical considerations in the approach. Despite being extremely tedious, positional cloning projects in the zebrafish will be comparatively easier to accomplish than in human and mouse, because of unique biological advantages of the zebrafish system. Moreover, the ease and speed at which genes are identified and cloned should rapidly increase as more mapping reagents and information become available, thereby paving the way for meaningful biological studies.

Animals↗
PubMed1998

Distinct patterns of IgH structure and organization in a divergent lineage of chrondrichthyan fishes.

Immunoglobulin heavy chain (IgH) genes in representative chondrichthyan fishes (sharks and skates) consist of independently functioning clusters, containing separate variable (VH), diversity (DH), and joining (JH) region elements and constant (CH) region exons. IgH loci have been characterized in Hydrolagus colliei (spotted ratfish), a modern representative of a major independent chondrichthyan lineage. Three distinct families of IgH gene clusters were identified. The most numerous genes consist of unjoined VH-D1-D2-JH segments that correspond to the most abundant Hydrolagus spleen (cDNA) transcripts which apparently arise from a diversified gene family. In the second cluster type, VH, DH, and JH segments are germline-joined, whereas the CH exons exhibit typical organization. This gene type is found in only a few copies per haploid genome and both transmembrane and secretory transcripts have been identified. A third cluster type has been identified that consists of unjoined VH elements but lacks a typical CH1 exon, which is substituted with a second CH2-like exon. Transcripts from this third cluster type also appear to derive from a diversified gene family. Genomic D regions of the two unjoined clone types exhibit structural differences that are consistent with incorporation of recombination machinery-mediated events. Genomic library screening indicates that 90% of VH+ clones are truncated, nearly identical pseudogenes (lacking JH and CH). These studies demonstrate an early phylogenetic origin for the cluster type of gene organization and document extensive organizational diversification within an apparent single class of IgH genes.

Amino Acid Sequence↗
PubMed1997

Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA.

We describe a simple electroelution method for purifying large, gel-fractionated DNA molecules that alleviates the need for melting of the agarose and subsequent enzymatic agarose digestion. The method yields DNA that is visibly more intact than that purified from a standard agarose-digestion protocol and is more amenable to large-fragment cloning with PAC and BAC vectors. These findings are notable in that PAC and BAC library construction is a very labor-intensive and costly procedure, such that any net improvement in cloning efficiency is highly advantageous. This method also should prove useful towards other applications which require purification of very large DNA molecules, such as YAC cloning.

Animals↗
PubMed1996

Human Txk: genomic organization, structure and contiguous physical linkage with the Tec gene.

Txk is a Tec-family tyrosine kinase expressed in mouse and human T lymphocytes. Among the Tec kinases, Txk is unique in that its amino terminal region does not include a pleckstrin homology domain or other known extended functional region. Txk is encoded at human chromosome 4p12 and at a recognized region of conserved synteny on mouse chromosome 5. The genomic organization of Txk consists of 15 exons with strong exon-intron organizational homology to Btk, the only other Tec-family kinase for which the genomic structure is fully known. The human Tec gene also maps to 4p12 and, based on limited studies reported here, possesses organizational homology with Btk and Txk. We have sequenced a continuous region of DNA that contains 3' Tec and 5' Txk exons separated by only a approximately 1.5 kb intergenic region containing the putative promoter region of Txk. The close physical linkage of these Tec-family tyrosine kinases, which are expressed in different hematopoetic cell lineages, suggests their potential for coordinate cis-regulation.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

A nonradioactive method for improved restriction analysis and fingerprinting of large P1 artificial chromosome clones.

The P1 artificial chromosome (PAC) cloning system is very useful for physical mapping, however, the large insert sizes cause difficulty in routine restriction analysis. In order to facilitate restriction mapping and fingerprinting, we have developed a simple, nonradioactive method for end-labeling and detection of restriction fragments from PAC clones. This method is very easy to implement, gives good differentiation of restriction fragments, and uses comparatively small amounts of DNA. We have used this method for restriction analysis of PAC clones containing inserts from human as well as from lower vertebrates. The method should also be applicable to other large-insert plasmid systems.

Animals↗
PubMed1994

A new bacteriophage P1-derived vector for the propagation of large human DNA fragments.

We have designed a P1 vector (pCYPAC-1) for the introduction of recombinant DNA into E. coli using electroporation procedures. The new cloning system, P1-derived artificial chromosomes (PACs), was used to establish an initial 15,000 clone library with an average insert size of 130-150 kilobase pairs (kb). No chimaerism has been observed in 34 clones, by fluorescence in situ hybridization. Similarly, no insert instability has been observed after extended culturing, for 20 clones. We conclude that the PAC cloning system will be useful in the mapping and detailed analysis of complex genomes.

Bacteriophage P1↗
PubMed1993

VH gene organization in a relict species, the coelacanth Latimeria chalumnae: evolutionary implications.

The living coelacanth Latimeria chalumnae is a relict species whose higher-level phylogenetic relationships have not been resolved clearly by traditional systematic approaches. Previous studies show that major differences in immunoglobulin gene structure and organization typify different phylogenetic lineages. To date, mammalian-, avian-, and elasmobranch-type gene organizations have been identified in representatives of these different phylads. A fourth form or organization is found in Latimeria, which possesses immunoglobulin heavy-chain variable region (VH) elements separated by approximately 190 nucleotides from diversity (D) elements. Adjacency of VH and D elements is characteristic of the elasmobranch "clustered" arrangement, although many other features of coelacanth VH gene organization and structure are more similar to those of bony fishes and tetrapods. These observations strongly support a phylogenetic hypothesis in which Latimeria occupies a sister-group relationship with teleosts and tetrapods.

Amino Acid Sequence↗